Plasma levels of coagulation factors VIII and IX and risk of venous thromboembolism: Systematic review and meta-analysis.

INTRODUCTION: The associations of plasma factor VIII (FVIII) and factor IX (FIX) levels with risk of venous thromboembolism (VTE) are not well defined. We performed a systematic review and meta-analysis of these associations. METHODS: Random effects inverse-variance weighted meta-analysis was used to estimate pooled odds ratios for comparisons across equal quartiles of the distributions and 90 % thresholds (higher versus lower), and for testing linear trends. RESULTS: Among 15 studies (5327 cases) the pooled odds ratio of VTE for the fourth versus first quarter was 3.92 (95 % confidence interval 1.61, 5.29) for FVIII level; and among 7 studies (3498 cases) 1.57 (1.32, 1.87) for FIX level. Comparing factor levels above, versus below, the 90th percentile, the estimated pooled odds ratios were 3.00 (2.10, 4.30) for FVIII; 1.77 (1.22, 2.56) for FIX; and 4.56 (2.73, 7.63) for both FVIII and FIX considered jointly. CONCLUSIONS: We confirm increases in risk of VTE across population distributions of FVIII and FIX levels. Levels above the 90th percentile have almost twice the risk for FIX level compared to levels below; three-fold risk for FVIII level; and almost five-fold risk for both FVIII and FIX levels elevated.

Plasma viscosity, immunoglobulins and risk of cardiovascular disease and mortality: new data and meta-analyses.

AIMS: Associations of plasma viscosity and plasma Ig levels (a determinant of viscosity) with incident coronary heart disease (CHD) events; and with CHD, cardiovascular disease (CVD: CHD and stroke) and all-cause mortalities. METHODS: Meta-analysis of plasma viscosity levels from the MONitoring of trends and determinants of CArdiovascular (MONICA)/Cooperative Health Research in the Region of Augsburg, MONICA Glasgow and Speedwell Studies; and five other published studies. Meta-analysis of IgA, IgG and IgM levels from the Augsburg, Glasgow and Speedwell studies; and one other published study. RESULTS: Over median follow-up periods of 14-26 years, there were 2270 CHD events, and 4220 all cause deaths in 28 605 participants with baseline plasma viscosity measurements. After adjustment for major risk factors, (HRs; 95% CIs) for a 1 SD increase in viscosity were 1.14 (1.09 to 1.20) for CHD events; and 1.21 (1.17 to 1.25) for all-cause mortality. 821 CHD events and 2085 all-cause deaths occurred in 8218 participants with baseline Ig levels. For CHD events, adjusted HRs for 1 SD increases in IgA, IgG and IgM were, respectively, 0.97 (0.89 to 1.05); 0.95(0.76 to 1.17) and 0.90 (0.79 to 1.03). Corresponding adjusted HRs for all-cause mortality were 1.08 (95% CI 1.02 to 1.13), 1.03 (95% CI 0.94 to 1.14) and 1.01 (95% CI 0.96 to 1.06). CONCLUSIONS: After risk factor adjustment, plasma viscosity was significantly associated with risks of CHD events; and with CHD, CVD and all-cause mortalities. We found no significant association of IgA, IgG or IgM levels with incident CHD events or mortality, except for a borderline association of IgA with all-cause mortality.

Thrombosis in Scotland, 1800-1960.

From 1800, arterial thrombosis and venous thrombosis were increasingly recognised as causes of sudden death. By 1960, they had replaced infections as the commonest cause of death in Scotland and the UK, as in many developed countries. The important contributions of Scotland's doctors between 1800 and 1960 to the knowledge of thrombosis, and to its treatment and prevention by anticoagulant drugs, are reviewed.

Haematological variables and risk of future venous thromboembolism in the British Regional Heart Study on men. Combined D-dimer and APTT as a predictive test for thromboembolism?

We examined the associations between haematological and inflammatory variables with future venous thromboembolism (VTE), in 3494 men aged 60-79 years, with no previous history of VTE or myocardial infarction, who were not receiving oral anticoagulants. After a mean follow-up period of 18 years, there were 149 confirmed cases of fatal or non-fatal VTE (deep vein thrombosis and/or pulmonary embolism). Among classical cardiovascular risk factors, only obesity and cigarette smoking were associated with VTE risk. After adjustment for age, obesity and smoking, VTE risk was associated with coagulation factor VIII, factor IX, von Willebrand factor (VWF), activated partial thromboplastin time (APTT), and fibrin D-dimer. Hazard ratios (95% CI) for top to bottom quarters (bottom to top for APTT), were respectively 2.17 (1.37, 3.44), 2.15 (1.30, 3.53), 2.02 (1.27, 3.22), 2.43 (1.47, 4.02) and 3.62 (2.18, 6.08). The 11% of men with both the shortest APTT and highest D-dimer combined had a 5.02 (2.37, 10.62) higher risk of VTE. VTE risk was not associated with fibrinogen, factor VII or activated protein C resistance; full blood count variables or with inflammatory markers, plasma viscosity, C-reactive protein or interleukin-6. The combination of D-dimer and APTT merits evaluation as an adjunct to VTE risk prediction scores.

Associations of Hemostatic Variables with Cardiovascular Disease and Total Mortality: The Glasgow MONICA Study.

The associations of plasma levels of hemostatic factors, other than fibrinogen, with risks of cardiovascular disease (CVD) and all-cause mortality are not well defined. In two phases of the Glasgow MONICA study, we assayed coagulation factors (VII, VIII, IX, and von Willebrand factor), coagulation inhibitors (antithrombin, protein C, protein S), coagulation activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin complexes, D-dimer), and the fibrinolytic factors, tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor type 1. Over 15 to 20 years, we followed up between 382 and 1,123 men and women aged 30 to 74 years, without baseline CVD, for risks of CVD and mortality. Age- and sex-adjusted hazard ratios (HRs) for CVD (top third vs bottom third) were significant only for factor VIII (1.30; 95% confidence interval [CI], 1.06-1.58) and factor IX (1.18; 95% CI, 1.01-1.39); these HRs were attenuated by further adjustment for CVD risk factors: 1.17 (95% CI, 0.94-1.46) and 1.07 (95% CI, 0.92-1.25), respectively. In contrast, factor VIII (HR, 1.63; 95% CI, 1.35-1.96), D-dimer (HR, 2.34; 95% CI, 1.26-4.35), and t-PA (HR, 2.81; 95% CI, 1.43-5.54) were strongly associated with mortality after full risk factor adjustment. Further studies, including meta-analyses, are required to assess the associations of these hemostatic factors with the risks of stroke and heart disease and causes of mortality.

An insight into anti-adipogenic properties of an Oroxylum indicum (L.) Kurz extract.

BACKGROUND: Oroxylum indicum fruit extract (OIE) has been reported to inhibit the development of adipocytes. However, the exact mechanism of its metabolic activity is not clearly defined. This study attempted to investigate whether OIE was involved in disrupting the cell cycle, glucose metabolism, and mitochondrial function in 3 T3-L1 cells. METHODS: The effect of the OIE on cell cycle progression was measured by flow cytometry along with observing the expression of the cycle regulator by immunoblotting. The effect of the OIE on glucose metabolism was investigated. The amount of glucose uptake (2-NBDG) influenced by insulin was determined as well as the protein tyrosine phosphorylation (PY20), and glucose transporter4 (GLUT4) expression was determined by immunoblotting assay. Mitochondria are also essential to metabolic processes. This study investigated mitochondrial activity using fluorescent lipophilic carbocyanine dye (JC-1) and mitochondria mass by MitoTracker Green (MTG) staining fluorescence dyes. Finally, cellular ATP concentration was measured using an ATP chemiluminescence assay. RESULTS: Treatment with OIE plus adipogenic stimulators for 24 h arrested cell cycle progression in the G2/M phase. Moreover, 200 µg/mL of OIE significantly diminished the expression of the insulin receptor (IR) and GLUT4 protein compared to the untreated-adipocytes (P < 0.05). The mitochondrial membrane potential (MMP) was significantly reduced (24 h) and increased (day 12) by OIE compared to untreated-adipocytes (P < 0.05). However, OIE maintained MMP and ATP at a similar level compared to the pre-adipocytes (day 12). Transmission electron microscope (TEM) results demonstrated that OIE could protect mitochondria deformation compared to the untreated-adipocytes. CONCLUSION: These results suggest that the inhibitory effect of the OIE on adipogenesis may potentially inhibit the cell cycle and phosphorylation of IR, leading to a decrease in glucose uptake to the cells. The OIE also slows down the mitochondrial activity of the early phase of cell differentiation, which can also inhibit the development of fat cells.

The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis).

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu2+), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH (P < 0.05; IC50PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18 µg/ml) and peroxynitrite scavenging assays. PIT at 75 µg/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0.05; IC50PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67 µg/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress.

History of the West of Scotland Haemophilia Centre, Glasgow, 1950-2019.

Over 70 years, the West of Scotland Haemophilia Centre in the UK has played a leading role in research, education and training. Its staff studied the natural history of haemophilias, their complications, and their treatment complications, pioneered the use of fibrinolytic inhibitors to reduce the risk of receiving a blood transfusion and developed national audit. Collaborations across Scotland with other haemophilia centres and the Scottish National Blood Transfusion Service progressed self-suffi ciency in NHS-produced factor concentrates, heat treatments to prevent HIV and hepatitis transmission, and finally, replacement of human by recombinant factor concentrates.

Pluchea indica (L.) Less. Tea Ameliorates Hyperglycemia, Dyslipidemia, and Obesity in High Fat Diet-Fed Mice.

Pluchea indica (L.) Less. (P. indica) tea has been used for a health-promoting drink, especially in Southeast Asia. The effect of P. indica tea (PIT) on amelioration of hyperglycemia; dyslipidemia that was total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), and triglyceride (TG); and obesity in high fat diet-induced (HFD) mice was investigated. Oral glucose tolerance test (OGTT) displayed that PIT at 400 and 600 mg/kg orally ameliorated hyperglycemia with a dose-dependent manner compared to the untreated group. Moreover, PIT at these dosages exhibited significantly lower TC, LDL-C, TG, and perigonadal fat weight in HFD treated mice compared to HFD mice (P < 0.05) with a dose-dependent manner. In contrast, HDL-C was higher than in the HFD group, but not a significant difference (P > 0.05). The PIT chemical analysis results demonstrated that PIT contained total phenolic content (TPC), total flavonoid content (TFC), 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), 4,5-O-dicaffeoylquinic acid (4,5-CQ), beta-caryophyllene, and gamma-gurjunene that may play an important role in inhibiting hyperlipidemia and hyperglycemia. Also, histological analysis expressed that the mean area and amount of perigonadal fat adipocytes of PIT treated groups were significantly lower and higher than the HFD group (P < 0.05), respectively. The toxicity test of PIT at 600 mg/kg/day in mice showed that serum creatinine, alanine transaminase (ALT), alkaline phosphatase (ALP), and complete blood count (CBC) levels of HFD and PIT treated groups were not significantly different compared to the normal control diet group (NCD) (P > 0.05). These results suggest that PIT does not become toxic to the kidney, liver, and blood. In conclusion, PIT has the potential to develop into healthy food supplement or medicine for the prevention and treatment of hyperglycemic, hyperlipidemic, and obese patients.

Antiadipogenesis of Oroxylum indicum (L.) Kurz Extract via PPARγ2 in 3T3-L1 Adipocytes.

Oroxylum indicum is regarded as a traditional food with medicinal properties and is used widely throughout Asia. It has previously been demonstrated that O. indicum extract (OIE) was able to suppress the differentiation of 3T3-L1 preadipocytes to adipocytes. However, the mechanism underlying the antiadipogenesis of this plant has not been fully investigated. The present study aimed to explore the impact of OIE at 50 to 200 µg mL-1 on the molecular mechanism involved in the antiadipogenic activity in 3T3-L1 cells at day 0 of their differentiation to adipocytes. The morphology and biochemistry of the cells on day 12 were investigated and compared to the relevant controls. Adiponectin was measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARγ2), sterol regulatory element-binding proteins 1c (SREBP-1c), fatty acid synthetase (FAS), glucose transporter (GLUT4), and leptin in adipocytes was determined by real-time PCR. The results demonstrated that the OIE at 200 µg mL-1 exhibited strongest suppression on intracellular lipid accumulation. The levels of adiponectin were dramatically increased in the untreated adipocytes, whereas significantly decreased in the 200 µg mL-1 OIE-treated adipocytes (P < 0.05). Expression of the mRNAs revealed that OIE-treated adipocytes at 200 µg mL-1 significantly inhibited the expression of PPARγ2 and SREBP-1c and lowered the level of expression of GLUT4, FAS, and leptin compared to the control (P < 0.05). These findings suggest that OIE inhibits adipocyte differentiation along with the downregulation of PPARγ2, SREBP-1c, and GLUT4, leading to the decrease in the expression of FAS and adipokine (leptin and adiponectin). Thus, OIE might be developed for hyperlipidemia and obesity prevention.

Reduced Carotenoid and Retinoid Concentrations and Altered Lycopene Isomer Ratio in Plasma of Atopic Dermatitis Patients.

Carotenoids and retinoids are known to alter the allergic response with important physiological roles in the skin and the immune system. In the human organism various carotenoids are present, some of which are retinoid precursors. The bioactive derivatives of these retinoids are the retinoic acids, which can potently activate nuclear hormone receptors such as the retinoic acid receptor and the retinoid X receptor. In this study, we aimed to assess how plasma carotenoid and retinoid concentrations along with the ratio of their isomers are altered in atopic dermatitis (AD) patients (n = 20) compared to healthy volunteers (HV, n = 20). The study indicated that plasma levels of the carotenoids lutein (HV 198 ± 14 ng/mL, AD 158 ± 12 ng/mL, p = 0.02; all values in mean ± SEM), zeaxanthin (HV 349 ± 30 ng/mL, AD 236 ± 18 ng/mL, p ≤ 0.01), as well as the retinoids retinol (HV 216 ± 20 ng/mL, AD 167 ± 17 ng/mL, p = 0.04) and all-trans-retinoic acid (HV 1.1 ± 0.1 ng/mL, AD 0.7 ± 0.1 ng/mL, p = 0.04) were significantly lower in the AD-patients, while lycopene isomers, α-carotene, and ß-carotene levels were comparable to that determined in the healthy volunteers. In addition, the ratios of 13-cis- vs. all-trans-lycopene (HV 0.31 ± 0.01, AD 0.45 ± 0.07, p = 0.03) as well as 13-cis- vs. all-trans-retinoic acid (HV 1.4 ± 0.2, AD 2.6 ± 0.6, p = 0.03) were increased in the plasma of AD-patients indicating an AD-specific 13-cis-isomerisation. A positive correlation with SCORAD was calculated with 13-cis- vs. all-trans-lycopene ratio (r = 0.40, p = 0.01), while a negative correlation was observed with zeaxanthin plasma levels (r = -0.42, p = 0.01). Based on our results, we conclude that in the plasma of AD-patients various carotenoids and retinoids are present at lower concentrations, while the ratio of selected lycopene isomers also differed in the AD-patient group. An increase in plasma isomers of both lycopene and retinoic acid may cause an altered activation of nuclear hormone receptor signaling pathways and thus may be partly responsible for the AD-phenotype.

The Effect of Pluchea indica (L.) Less. Tea on Adipogenesis in 3T3-L1 Adipocytes and Lipase Activity.

Obesity and hyperlipidemia are a major problem in the world. Pluchea indica (L.) Less. tea (PIT) is a beverage that has various indications. This study focused on the effect of the PIT on inhibiting adipogenesis of 3T3-L1 cells and pancreatic lipase enzyme activity. The viability of 3T3-L1 cells was not significantly decreased after exposure to 200 to 1000 µg mL-1 PIT compared to controls (p > 0.05). The PIT at 750 to 1000 µg mL-1 exhibited a significantly reduced lipid accumulation compared to the control (p < 0.05). The inhibitory effects of the PIT at 250 to 1000 µg mL-1 on lipase activity were significantly increased compared to control (p < 0.05). The FTIR results showed that the integrated areas of lipids, proteins, nucleic acids, glycogen, and carbohydrates of the PIT-treated 3T3-L1 adipocytes were significantly lower than the untreated 3T3-L1 adipocytes (p < 0.05). These findings may indicate that the PIT is not only capable of inhibiting lipids and carbohydrate accumulation in adipocytes but also has a potential to inhibit pancreatic lipase activity. So, the PIT may be further developed to the novel lipid-lowering herbal supplement for the management of overweight or obesity.

Oroxylum indicum (L.) Kurz extract inhibits adipogenesis and lipase activity in vitro.

BACKGROUND: Oroxylum indicum (L.) Kurz (O. indicum) is found in Thailand. It has been used for the treatment of obesity. This study aimed to investigate the effects of an O. indicum extract (OIE) on the adipogenic and biomolecular change in 3T3-L1 adipocytes. METHODS: Initial studies examined the chemical components of OIE. The cell line 3T3-L1 was used to establish potential toxic effects of OIE during the differentiation of pre-adipocytes to adipocytes. The inhibitory effect of OIE on lipid accumulation in 3T3-L1 cells was investigated. Moreover, the impact of OIE on pancreatic lipase activity was determined. In further experiments, Fourier Transform Infrared (FTIR) was used to monitor and discriminate biomolecular changes caused by the potential anti-adipogenic effect of OIE on 3T3-L1 cells. RESULTS: Chemical screening methods indicated that OIE was composed of flavonoids, alkaloids, steroids, glycosides, and tannins. The percentage viability of 3T3-L1 cells was not significantly decreased after exposure to either 200 or 150 µg/mL of OIE for 2 and 10 days, respectively compared to control cells. The OIE exhibited a dose-dependent reduction of lipid accumulation compared to the control (p < 0.05). The extract also demonstrated a dose-dependent inhibitory effect upon lipase activity compared to the control. The inhibitory effect of the OIE on lipid accumulation in 3T3-L1 cells was also confirmed using FTIR microspectroscopy. The signal intensity and the integrated areas relating to lipids, lipid esters, nucleic acids, glycogen and carbohydrates of the OIE-treated 3T3-L1 adipocytes were significantly lower than the non-treated 3T3-L1 adipocytes (p < 0.05). Principal component analysis (PCA) indicated four distinct clusters for the FTIR spectra of 3T3-L1 adipocytes based on biomolecular changes (lipids, proteins, nucleic acids, and carbohydrates). This observation was confirmed using Unsupervised hierarchical cluster analysis (UHCA). CONCLUSIONS: These novel findings provide evidence that the OIE derived from the fruit pods of the plant is capable of inhibiting lipid and carbohydrate accumulation in adipocytes and also has the potential to inhibit an enzyme associated with fat absorption. The initial observations indicate that OIE may have important properties which in the future may be exploited for the management of the overweight or obese.

Troponin and BNP are markers for subsequent non-ischaemic congestive heart failure: the Caerphilly Prospective Study (CaPS).

Objective: To examine the long-term predictive value of 28 biomarkers for subsequent non-ischaemic congestive heart failure (CHF) and separately for other cardiovascular outcomes (myocardial infarction (MI) and stroke). Methods: The Caerphilly Prospective Study recruited 2171 men aged 55-69 years from the general population in 1989-1993; men were screened for evidence of cardiovascular disease (CVD) and followed for clinical cardiovascular events. Fasting blood samples were stored at -70°C until assayed for novel biomarkers in 2010-2013. A competing risks proportional hazards regression analysis was used to estimate subhazard ratios (SHRs) for each biomarker for each cardiovascular outcome. Results: During follow-up (average 13 years), only new, initial events were evaluated in the whole cohort: 584 MIs, 313 strokes and 261 episodes of CHF (not associated with acute MI). In a subcohort of men who had no clinical history or evidence of CVD at baseline examination (n=1279) those in the top third of the distributions of troponin and B-type natriuretic peptide (BNP) showed a threefold increase in risk for subsequent CHF as a first event after adjustment for all conventional risk factors (SHRs 3.37, 95% CI 1.39 to 8.14 and 3.23, 95% CI 1.45 to 7.23), respectively, in contrast to moderate elevations in risk for acute MI (troponin SHR 1.63, 95% CI 1.10 to 2.41) and for stroke (BNP SHR 1.75 95% CI 1.06 to 2.88). Conclusion: Troponin and BNP could be considered as potentially useful screening tools to detect subjects without prior CVD at increased risk of developing CHF in subsequent years in addition to having lesser roles for predicting subsequent MI (troponin) or stroke (BNP).

Carotenoids-Antioxidant Properties.

The carotenoid group of pigments are ubiquitous in nature and more than 600 different carotenoids have been identified and characterized [1].[...].

Associations of time of day with cardiovascular disease risk factors measured in older men: results from the British Regional Heart Study.

OBJECTIVE: We estimated associations of time of day with cardiovascular disease (CVD) risk factors measured in older men. METHODS: CVD risk factors (markers of inflammation and haemostasis, and cardiac markers) were measured on one occasion between 08:00 and 19:00 hours in 4252 men aged 60-79 years from the British Regional Heart Study. Linear models were used to estimate associations between time of day and risk factors. When an association was found, we examined whether the relationship between risk factors and cardiovascular mortality was affected by the adjustment for time of day using survival analyses. RESULTS: N-terminal pro-brain natriuretic peptide (NT-proBNP) levels increased by 3.3% per hour (95% CI 1.9% to 4.8%), interleukin-6 (IL-6) increased by 2.6% per hour (95% CI 1.8% to 3.4%), while tissue plasminogen activator (t-PA) decreased by 3.3% per hour (95% CI 3.7% to 2.9%); these associations were unaffected by adjustment for possible confounding factors. The percentages of variation in these risk factors attributable to time of day were less than 2%. In survival analyses, the association of IL-6, NT-proBNP and t-PA with cardiovascular mortality was not affected by the adjustment for time of day. C reactive protein, fibrinogen, D-dimer, von Willebrand factor and cardiac troponin T showed no associations with time of day. CONCLUSIONS: In older men, markers of inflammation (IL-6), haemostasis (t-PA) and a cardiac marker (NT-proBNP) varied by time of day. The contribution of time of day to variations in these markers was small and did not appear to be relevant for the CVD risk prediction.